Compressive mechanical force augments osteoclastogenesis by bone marrow macrophages through activation of c‐Fms‐mediated signaling

Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)‐α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage‐colony stimulating factor (M‐CSF) and is further facilitated by receptor activator of nuclear factor‐kappaB (NF‐κB) ligand (RANKL). Application of force to BMMs accelerated TNF‐α‐induced osteoclastogenesis; this was inhibited either by anti‐TNF‐α or anti‐TNF‐α receptor but not by OPG. TNF‐α also increased c‐Fms expression at both mRNA and protein levels in BMMs. An anti‐c‐Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF‐α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF‐α (in the presence of M‐CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c‐Fms‐mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells. J. Cell. Biochem. 111: 1260–1269, 2010. © 2010 Wiley‐Liss, Inc.     

Caffeic acid phenethyl ester,an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley‐Liss, Inc.     

Stachydrine prevents LPS‐induced bone loss by inhibiting osteoclastogenesis via NF‐κB and Akt signalling

Osteoclast overactivation‐induced imbalance in bone remodelling leads to pathological bone destruction, which is a characteristic of many osteolytic diseases such as rheumatoid arthritis, osteoporosis, periprosthetic osteolysis and periodontitis. Natural compounds that suppress osteoclast formation and function have therapeutic potential for treating these diseases. Stachydrine (STA) is a bioactive alkaloid isolated from Leonurus heterophyllus Sweet and possesses antioxidant, anti‐inflammatory, anticancer and cardioprotective properties. However, its effects on osteoclast formation and function have been rarely described. In the present study, we found that STA suppressed receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL)‐induced osteoclast formation and bone resorption, and reduced osteoclast‐related gene expression in vitro. Mechanistically, STA inhibited RANKL‐induced activation of NF‐κB and Akt signalling, thus suppressing nuclear factor of activated T cells c1 induction and nuclear translocation. In addition, STA alleviated bone loss and reduced osteoclast number in a murine model of LPS‐induced inflammatory bone loss. STA also inhibited the activities of NF‐κB and NFATc1 in vivo. Together, these results suggest that STA effectively inhibits osteoclastogenesis both in vitro and in vivo and therefore is a potential option for treating osteoclast‐related diseases.     

Dehydrocostus lactone attenuates osteoclastogenesis and osteoclast‐induced bone loss by modulating NF‐κB signalling pathway

Osteolysis is characterized by overactivated osteoclast formation and potent bone resorption. It is enhanced in many osteoclast‐related diseases including osteoporosis and periprosthetic osteolysis. The shortage of effective treatments for these pathological processes emphasizes the importance of screening and identifying potential regimens that could attenuate the formation and function of osteoclasts. Dehydrocostus lactone (DHE) is a natural sesquiterpene lactone containing anti‐inflammatory properties. Here, we showed that DHE suppressed receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclast formation and osteoclast marker gene expression. It also inhibited F‐actin ring formation and bone resorption in a dose‐dependent manner in vitro. Moreover, DHE inhibited the RANKL‐induced phosphorylation of NF‐κB, mitigated bone erosion in vivo in lipopolysaccharide‐induced inflammatory bone loss model and particle‐induced calvarial osteolysis model. Together, these results suggest that DHE reduces osteoclast‐related bone loss via the modulation of NF‐κB activation during osteoclastogenesis indicating that it might be a useful treatment for osteoclast‐related skeletal disorders.     

Mangiferin attenuates osteoclastogenesis,bone resorption,and RANKL‐induced activation of NF‐κB and ERK

Osteolytic bone diseases such as osteoporosis have a common pathological feature in which osteoclastic bone resorption outstrips bone synthesis. Osteoclast formation and activation are regulated by receptor activator of nuclear factor κB ligand (RANKL). The induction of RANKL‐signaling pathways occurs following the interaction of RANKL to its cognate receptor, RANK. This specific binding drives the activation of downstream signaling pathways; which ultimately induce the formation and activation of osteoclasts. In this study, we showed that a natural immunomodulator, mangiferin, inhibits osteoclast formation and bone resorption by attenuating RANKL‐induced signaling. Mangiferin diminished the expression of osteoclast marker genes, including cathepsin K, calcitonin receptor, DC‐STAMP, and V‐ATPase d2. Mechanistic studies revealed that mangiferin inhibits RANKL‐induced activation of NF‐κB, concomitant with the inhibition of IκB‐α degradation, and p65 nuclear translocation. In addition, mangiferin also exhibited an inhibitory effect on RANKL‐induced ERK phosphorylation. Collectively, our data demonstrates that mangiferin exhibits anti‐resorptive properties, suggesting the potential application of mangiferin for the treatment and prevention of bone diseases involving excessive osteoclastic bone resorption. J. Cell. Biochem. 112: 89–97, 2011. © 2010 Wiley‐Liss, Inc.     

Receptor activator of nuclear factor‐kappa B ligand induces osteoclast formation in RAW 264.7 macrophage cells via augmented production of macrophage–colony‐stimulating factor

RAW 264.7 macrophage cells differentiate into osteoclast‐like cells in the presence of RANKL. Participation of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells was examined. TRAP‐positive osteoclast‐like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL‐induced osteoclast formation was markedly inhibited by anti‐M‐CSF antibody. RANKL augmented M‐CSF mRNA expression and M‐CSF production in RAW 264.7 cells. Further, anti‐M‐CSF antibody inhibited the expression of RANK, c‐fms, c‐fos and TRAP mRNA in RANKL‐stimulated RAW 264.7 cells. However, anti‐M‐CSF antibody did not affect the expression of DC‐STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M‐CSF. The putative role of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells is discussed.     

Oxymatrine exerts protective effects on osteoarthritis via modulating chondrocyte homoeostasis and suppressing osteoclastogenesis

Osteoarthritis (OA) is a common degenerative disease characterized by the progressive destruction both articular cartilage and the subchondral bone. The agents that can effectively suppress chondrocyte degradation and subchondral bone loss are crucial for the prevention and treatment of OA. Oxymatrine (OMT) is a natural compound with anti‐inflammatory and antitumour properties. We found that OMT exhibited a strong inhibitory effect on LPS‐induced chondrocyte inflammation and catabolism. To further support our results, fresh human cartilage explants were treated with LPS to establish an ex vivo degradation model, and the results revealed that OMT inhibited the catabolic events of LPS‐stimulated human cartilage and substantially attenuated the degradation of articular cartilage ex vivo. As subchondral bone remodelling is involved in OA progression, and osteoclasts are a unique cell type in bone resorption, we investigated the effects of OMT on osteoclastogenesis, and the results demonstrated that OMT suppresses RANKL‐induced osteoclastogenesis by suppressing the RANKL‐induced NFATc1 and c‐fos signalling pathway in vitro. Further, we found that the anti‐inflammatory and anti‐osteoclastic effects of oxymatrine are mediated via the inhibition of the NF‐κB and MAPK pathways. In animal studies, OMT suppressed the ACLT‐induced cartilage degradation, and TUNEL assays further confirmed the protective effect of OMT on chondrocyte apoptosis. MicroCT analysis revealed that OMT had an attenuating effect on ACLT‐induced subchondral bone loss in vivo. Taken together, these results show that OMT interferes with the vicious cycle associated with OA and may be a potential therapeutic agent for abnormal subchondral bone loss and cartilage degradation in osteoarthritis.     

Glycosaminoglycans modulate RANKL‐induced osteoclastogenesis

Skeletal integrity is tightly regulated by the activity of osteoblasts and osteoclasts that are both under the control of extracellular glycosaminoglycans (GAGs) through their interactions with endogenous growth factors and differentiation‐promoting ligands. Receptor activator of NF‐kappa‐B ligand (RANKL), which is a tumor necrosis factor (TNF)‐related protein that is critical for osteoclast formation, is produced by osteoblasts and further modulated by certain types of GAGs. Using unfractionated osteoblast‐derived GAGs that reflect the complex tissue microenvironment within which osteoclasts reside, we demonstrate that these GAGs block the osteoclastogenic activity of RANKL. Furthermore, RANKL significantly reduces extracellular signal‐regulated protein kinase (ERK) activity, a putative suppressor of osteoclastogenesis, but osteoblast‐derived GAGs eliminate the inhibitory effects of RANKL on ERK activity. Notably, while imposing an anti‐osteoclastic effect, these GAGs also enhanced the proliferation of osteoblasts. Thus, the osteoblast microenvironment is a potent source of GAGs that promote bone anabolic activities. The anti‐osteoclastogenic and osteoblast‐related mitogenic activities of these GAGs together may provide a key starting point for the development of selective sugar‐based therapeutic compounds for the treatment of osteopenic disorders. J. Cell. Biochem. 109: 1222–1231, 2010. © 2010 Wiley‐Liss, Inc.     

Artesunate inhibits RANKL‐induced osteoclastogenesis and bone resorption in vitro and prevents LPS‐induced bone loss in vivo

Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side‐effects caused by the currently available drugs, a continuous search for novel bone‐protective therapies is essential. Artesunate (Art), the water‐soluble derivative of artemisinin has been investigated owing to its anti‐malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, the mRNA expression of osteoclastic‐specific genes, and resorption pit formation in a dose‐dependent manner in primary bone marrow‐derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL‐induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF‐κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)‐induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL‐induced osteoclastogenesis by suppressing the NF‐κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases.     

Inhibition of PFKFB3 suppresses osteoclastogenesis and prevents ovariectomy‐induced bone loss

Osteoclasts are multinucleated cells derived from the monocyte/macrophage cell lineage under the regulation of receptor activator of nuclear factor‐κB ligand (RANKL). In previous studies, stimulation by RANKL during osteoclastogenesis was shown to induce a metabolic switch to enhanced glycolytic metabolism. Thus, we hypothesized that blockage of glycolysis might serve as a novel strategy to treat osteoclast‐related diseases. In the present study, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase 3 (PFKFB3), an essential regulator of glycolysis, was up‐regulated during osteoclast differentiation. Genetic and pharmacological inhibition of PFKFB3 in bone marrow‐derived macrophages suppressed the differentiation and function of osteoclasts. Moreover, intraperitoneal administration of the PFKFB3 inhibitor PFK15 prevented ovariectomy‐induced bone loss. In addition, glycolytic activity characterized by lactate accumulation and glucose consumption in growth medium was reduced by PFKFB3 inhibition. Further investigation indicated that the administration of L‐lactate partially reversed the repression of osteoclastogenesis caused by PFKFB3 inhibition and abrogated the inhibitory effect of PFK15 on the activation of NF‐κB and MAPK pathways. In conclusion, the results of this study suggest that blockage of glycolysis by targeting PFKFB3 represents a potential therapeutic strategy for osteoclast‐related disorders.     

Flesh‐eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor‐κB ligand

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock‐like syndrome. Human CD46‐expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast‐mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 10⁵ colony‐forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor‐κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro‐computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4⁺ and CD8⁺ T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte‐derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast‐like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.     

Inflammatory osteoclastogenesis can be induced by GM-CSF and activated under TNF immunity

In inflammatory arthritis such as RA, osteoclastic activity is severely enhanced. GM-CSF was reportedly elevated in synovial fluid, but is a strong inhibitor of osteoclastogenesis; here lies a contradiction. Our objective was to examine what type of osteoclasts generate and resorb bone with resistance to GM-CSF in an inflammatory joint. Monocyte-derived cells generated in GM-CSF were morphologically and immunophenotypically different from both the conventional DC and macrophage. They could differentiate into osteoclasts in the presence of RANKL + M-CSF, acquiring a stronger osteoclastic activity under TNF treatment. Furthermore, their differentiation was not inhibited by GM-CSF, while monocyte-derived osteoclast differentiation was completely inhibited. The resorption was suppressed by GM-CSF, and the existence of another osteoclastic pathway has been suggested. Our findings indicate another type of osteoclast exists in inflammatory arthritis.     

Glutathione accelerates osteoclast differentiation and inflammatory bone destruction

Chronic inflammation associated with bone tissues often destructs bones, which is essentially performed by osteoclasts in the presence of immunoregulatory molecules. Hence, regulating osteoclastogenesis is crucial to develop therapeutics for bone-destructive inflammatory diseases. It is believed that reactive oxygen species (ROS) are involved in receptor activator of NF-κB (RANK) ligand (RANKL)-induced osteoclast differentiation, and, therefore, glutathione (GSH), the most abundant endogenous antioxidant, suppresses osteoclast differentiation and bone resorption by RANKL. Interestingly, GSH also contributes to inflammatory responses, and the effects of GSH on osteoclast differentiation and bone destruction under inflammatory conditions have not yet been determined. Here, we investigated how GSH affects inflammatory cytokine-stimulated osteoclast differentiation in vitro and in a mouse model of inflammatory bone destruction. We found that GSH significantly promoted TNFα-stimulated osteoclast formation, while an inhibitor of GSH synthesis, buthionine sulfoximine, suppressed it. GSH facilitated the nuclear localisation of the nuclear factor of activated T cells c1 (NFATc1) protein, a master regulator of osteoclastogenesis, as well as the expression of osteoclast marker genes in a dose-dependent manner. N-acetylcysteine, a substrate of GSH synthesis, also stimulated osteoclast formation and NFATc1 nuclear localisation. GSH did not suppress cell death after osteoclast differentiation. In mouse calvaria injected with lipopolysaccharide, GSH treatment resulted in a fivefold increase in the osteolytic lesion area. These results indicate that GSH accelerates osteoclast differentiation and inflammatory bone destruction, suggesting GSH appears to be an important molecule in the mechanisms responsible for inflammatory bone destruction by osteoclasts.